Herpes simplex virus-2 (HSV-2) is one of the most common sexually transmitted infections (STIs) with a prevalence of 45 million in the USA. HSV-2 is primarily transmitted via exposure at the genital mucosal surfaces, which leads to the establishment of latency in the sacral ganglia. Although pharmacological interventions exist for HSV-2-related symptoms, there are no preventative vaccines or curative measures available for this disease. Towards developing vaccines to prevent HSV-2 transmission, a clear understanding of the mechanism by which immune responses are mediated within the relevant mucosal sites is necessary. Currently, the immune mechanisms of protection within the female genital mucosa are poorly understood. Work supported by the previous funding periods of this award led to the understanding that memory T cell circulation to the vagina is restricted, and requires CD4 T-dependent induction of chemokines in recruitment of effector CD8 T cells. Based on this, we developed a new vaccine strategy, Prime and Pull, in which topical application of chemokines following parenteral vaccination establishes local memory CD8 T cells to dramatically improve protection afforded by parenteral vaccines. Our Preliminary Studies indicate an establishment of long-lived tissue resident memory CD4 T cells following intravaginal immunization with TK- HSV-2. Tissue-resident memory CD4 T cells are maintained for life within memory lymphocyte clusters (MLC) that form beneath the vaginal epithelia layer. Moreover, by using parabiotic mice, we observe a striking reliance on the tissue-resident CD4 T cell memory population in protecting the host against a lethal HSV-2 challenge. Based on these data, the goal of this grant application is to understand the mechanism by which protective immunity against HSV-2 is orchestrated by local memory T cells in the female genital tract, and to apply this understanding to design a robust vaccine against genital herpes. We will probe the mechanism by which tissue-resident memory T cells are established and maintained in the genital mucosa (Aim 1), and to determine the how the secretion of chemokine that retain these T cells are maintained long terms (Aim 2). We will design a novel vaccine strategy based on the knowledge gained by the first two Aims (Aim 3). These studies will help to establish critical foundation with which to design immunological interventions and preventative measures against genital herpes and other deleterious STI diseases in humans.